Mutant luciferase

ABSTRACT

An isolated recombinant luciferase having luciferase activity. The recombinant luciferase has an amino acid sequence which differs from the wild-type luciferase from  Photinus pyralis, Luciola mingrelica, Luciola cruciata, Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyris noctiluca, Pyrocoelia miyako  or  Photinus pennsylvanica . In the sequence of the recombinant luciferase, the amino acid residue corresponding to phenylalanine 295 in  Photinus pyralis  wild-type luciferase or to leucine 297 in  Luciola mingrelica, Luciola cruciata  or  Luciola lateralis  wild-type luciferases, is mutated compared to the corresponding amino acid which appears in the corresponding wild-type luciferase sequence. The recombinant luciferase has increased thermostability compared to the corresponding wild-type luciferase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/763,824, filed Feb. 27, 2001, which is a national stage filing under35 USC 371 of International Application No. PCT/GB99/03538, filed Oct.26, 1999, which claims priority benefits to United Kingdom PatentApplication No. 9823468.5, filed Oct. 28, 1998. These applications areincorporated herein by reference in their entirety.

SUMMARY

The present invention relates to novel proteins, in particular mutantluciferase enzymes having increased thermostability as compared to thecorresponding wild type enzyme, to the use of these enzymes in assaysand to test kits containing them.

Firefly luciferase catalyses the oxidation of luciferin in the presenceof ATP, Mg²⁺ and molecular oxygen with the resultant production oflight. This reaction has a quantum yield of about 0.88. The lightemitting property has led to its use in a wide variety of luminometricassays where ATP levels are being measured. Examples of such assaysinclude those which are based upon the described in EP-B-680515 and WO96/02665.

Luciferase is obtainable directly from the bodies of insects, inparticular beetles such as fireflies or glow-worms. Particular speciesfrom which luciferases have been obtained include the Japanese GENJI orKEIKE fireflies, Luciola cruciata and Luciola lateralis, the EastEuropean firefly Luciola mingrelica, the North American firefly Photinuspyralis and the glow-worm Lampyris noctiluca. Other species from whichluciferase can be obtained are listed in Ye et al., Biochimica etBiophysica Acta, 1339 (1997) 39-52. Yet a further species is Phrixothrix(railroad-worms), as described by Viviani et al., Biochemistry, 38,(1999) 8271-8279.

However, since many of the genes encoding these enzymes have been clonedand sequenced, they may also be produced using recombinant DNAtechnology. Recombinant DNA sequences encoding the enzymes are used totransform microorganisms such as E. coli which then express the desiredenzyme product.

The heat stability of wild and recombinant type luciferases is such thatthey lose activity quite rapidly when exposed to temperatures in excessof about 30° C., particularly over 35° C. This instability causesproblems when the enzyme is used or stored at high ambient temperature,or if the assay is effected under high temperature reaction conditions,for example in order to increase reaction rate.

Mutant luciferases having increased thermostability are known fromEP-A-524448 and WO95/25798. The first of these describes a mutantluciferase having a mutation at position 217 in the Japanese fireflyluciferase, in particular by replacing a threonine residue with anisoleucine residue. The latter describes mutant luciferases having over60% similarity to luciferase from Photinus pyralis, Luciola mingrelica,Luciola cruciata or Luciola lateralis but in which the amino acidresidue corresponding to residue 354 of Photinus pyralis or 356 of theLuciola species is mutated such that it is other than glutamate.

The applicants have found yet further mutants which can bring aboutincreased thermostability and which may complement the mutations alreadyknown in the art.

The present invention provides a protein having luciferase activity andat least 60% similarity to luciferase from Photinus pyralis, Luciolamingrelica, Luciola cruciata or Luciola lateralis, Hotaria paroula,Pyrophorus plagiophthalamus Lampyris noctiluca, Pyrocoelia nayako,Photinus pennsylanvanica or Phrixothrix, wherein in the sequence of theenzyme, at least one of

(a) the amino acid residue corresponding to residue 214 in Photinuspyralis luciferase or to residue 216 of Luciola mingrelica, Luciolacruciata or Luciola lateralis luciferase;(b) the amino acid residue corresponding to residue 232 in Photinuspyralis luciferase or to residue 234 of Luciola mingrelica, Luciolacruciata or Luciola lateralis luciferase;(c) the amino acid residue corresponding to residue 295 in Photinuspyralis luciferase or to residue 297 of Luciola mingrelica, Luciolacruciata or Luciola lateralis luciferase;(d) the amino acid residue corresponding to amino acid 14 of thePhotinus pyralis luciferase or to residue 16 of Luciola mingrelica, 6residue 17 of Luciola cruciata or Luciola lateralis;(e) the amino acid residue corresponding to amino acid 35 of thePhotinus pyrails luciferase or to residue 37 of Luciola mingrelica 38 ofLuciola cruciata or Luciola lateralis;(f) the amino acid residue corresponding to amino acid residue 105 ofthe Photinus pyralis luciferase or to residue 106 of Luciola mingrelica,107 of Luciola cruciata or Luciola lateralis or 108 of Luciola lateralisgene;(g) the amino acid residue corresponding to amino acid residue 234 ofthe Photinus pyralis luciferase or to residue 236 of Luciola mingrelica,Luciola cruciata or Luciola lateralis;(h) the amino acid residue corresponding to amino acid residue 420 ofthe Photinus pyralis luciferase or to residue 422 of Luciola mingrelica,Luciola cruciata or Luciola lateralis;(i) the amino acid residue corresponding to amino acid residue 310 ofthe Photinus pyralis luciferase or to residue 312 of Luciola mingrelica,Luciola cruciata or Luciola lateralis; is different to the amino acidwhich appears in the corresponding wild type sequence and wherein theluciferase enzyme possesses has increased thermostability as compared toan enzyme having the amino acid of the corresponding wild-typeluciferase of a particular species at this position.

Preferably, the protein has luciferase activity and at least 60%similarity to luciferase from Photinus pyralis, Luciola mingrelica,Luciola cruciata or Luciola lateralis, Hotaria paroula, Pyrophorusplagiophthalamus Lampyris noctiluca, Pyrocoelia nayako, or Photinuspennsylanvanica.

In particular, the protein is a recombinant protein which has luciferaseactivity and substantially the sequence of a wild-type luciferase, forexample of Photinus pyralis, Luciola mingrelica, Luciola cruciata orLuciola lateralis, Hotaria paroula, Pyrophorus plagiophthalamus(Green-Luc GR), Pyrophorus plagiophthalamus (Yellow-Green Luc YG),Pyrophorus plaglophthalamus (Yellow-Luc YE), Pyrophorus plagiophthalamus

(Orange-Luc OR), Lampyris noctiluca, Pyrocelia nayako Photinuspennsylanvanica LY, Photinus pennsylanvanica KW, Photinuspennsylanvanica J19, or Phrixothrix green (Pv_(GR)) or red (Ph_(RE)) butwhich may include one or more, for example up to 100 amino acidresidues, preferably no more than 50 amino acids and more preferably nomore than 30 amino acids, which have been engineered to be different tothat of the wild type enzyme.

In particular, bioluminescent enzymes from species that can use thesubstrate D-luciferin(4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazole carboxylic acid)to produce light emission may form the basis of the mutant enzymes ofthe invention.

By way of example, where the protein has substantially the sequence ofluciferase of Photinus pyralis, in accordance with the invention, atleast one of

(a) the amino acid residue corresponding to residue 214 in Photinuspyralis luciferase has been changed to be other than threonine;(b) the amino acid residue corresponding to residue 232 in Photinuspyralis luciferase has been changed to be other than isoleucine;(c) the amino acid residue corresponding to residue 295 in Photinuspyralis luciferase has been changed to be other than phenylalanine;(d) the amino acid residue corresponding to amino acid 14 of thePhotinus pyralis luciferase has been changed to be other thanphenylalanine;(e) the amino acid residue corresponding to amino acid 35 of thePhotinus pyralis luciferase has been changed to be other than leucine;(f) the amino acid residue corresponding to amino acid residue 105 ofthe Photinus pyralis luciferase has been changed to be other thanalanine;(g) the amino acid residue corresponding to amino acid residue 234 ofthe Photinus pyralis luciferase has been changed to be other thanaspartic acid;(h) the amino acid residue corresponding to amino acid residue 420 ofthe Photinus pyralis luciferase has been changed to be other thanserine;(i) the amino acid residue corresponding to amino acid residue 310 ofthe Photinus pyralis luciferase has been changed to be other thanhistidine.

Where the protein has substantially the sequence of Luciola mingrelica,Luciola cruciata or Luciola lateralis enzyme, in accordance with theinvention, at least one of

(a) the amino acid residue corresponding to residue 216 of Luciolamingrelica, Luciola cruciata or Luciola lateralis luciferase is otherthan glycine (for Luciola mingrelica based sequences) or aparagine (forLuciola cruciata or Luciola lateralis) based sequences;(b) the amino acid residue corresponding to residue 234 of Luciolamingrelica, Luciola cruciata or Luciola lateralis luciferase is otherthan serine;(c) amino acid residue corresponding to residue 297 of Luciolamingrelica, Luciola cruciata or Luciola lateralis luciferase is otherthan leucine;(d) amino acid residue corresponding to amino acid 16 of Luciolamingrelica, or to amino acid 17 of Luciola cruciata or Luciola lateralisis other than phenylalanine;(e) amino acid residue corresponding to residue 37 of Luciolamingrelica, or 38 of Luciola cruciata or Luciola lateralis is other thanlysine;(f) amino acid residue corresponding to amino acid residue 106 ofLuciola mingrelica, or to amino acid 107 of Luciola cruciata or Luciolalateralis or to amino acid 108 of Luciola lateralis gene is other thanglycine;(g) amino acid residue corresponding to amino acid residue 236 ofLuciola mingrelica, Luciola cruciata or Luciola lateralis is other thanglycine;(h) amino acid residue corresponding to residue 422 of Luciolamingrelica, Luciola cruciata or Luciola lateralis is other thanthreonine;(i) amino acid residue corresponding to amino acid residue 312 ofLuciola mingrelica, Luciola cruciata or Luciola lateralis is other thanthreonine (for Luciola mingrelica based sequences) or valine (forLuciola cruciata or Luciola lateralis) based sequences.

The particular substituted amino acids in any case which give rise toenhanced thermostability can be determined by routine methods asillustrated hereinafter. In each case, different substitutions mayresult in enhanced thermostability. Substitution may be effected bysite-directed mutagenesis of DNA encoding native or suitable mutantproteins as would be understood by the skilled person. The invention inthis case is associated with the identification of the positions whichare associated with thermostability.

In general however, it may be desirable to consider substituting anamino acid of different properties to the wild type amino acid. Thushydrophilic amino acid residues may, in some cases be preferablysubstituted with hydrophobic amino acid residues and vice versa.Similarly, acidic amino acid residues may be substituted with basicresidues.

For instance, the protein may comprise a protein having luciferaseactivity and at least 60% similarity to luciferase from Photinuspyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralisenzyme wherein in the sequence of the enzyme, at least one of

(a) the amino acid residue corresponding to residue 214 in Photinuspyralis luciferase and to residue 216 of Luciola mingrelica, Luciolacruciata or Luciola lateralis luciferase is mutated and is other thanthreonine in the case of Photinus pyralis luciferase; or(b) the amino acid residue corresponding to residue 232 in Photinuspyralis luciferase and to residue 234 of Luciola mingrelica, Luciolacruciata or Luciola lateralis luciferase is mutated and is other thanisoleucine in the case of Photinus pyralis luciferase; or(c) amino acid residue corresponding to residue 295 in Photinus pyralisluciferase and to residue 297 of Luciola mingrelica, Luciola cruciata orLuciola lateralis luciferase is mutated and is for example, other thanphenylalanine in the case of Photinus pyralis luciferase;and the luciferase enzyme has increased thermostability as compared tothe wild-type luciferase.

The sequences of all the various luciferases show that they are highlyconserved having a significant degree of similarity between them. Thismeans that corresponding regions among the enzyme sequences are readilydeterminable by examination of the sequences to detect the most similarregions, although if necessary commercially available software (e.g.“Bestfit” from the University of Wisconsin Genetics Computer Group; seeDevereux et al (1984) Nucleic Acid Research 12: 387-395) can be used inorder to determine corresponding regions or particular amino acidsbetween the various sequences. Alternatively or additionally,corresponding acids can be determined by reference to L. Ye et al.,Biochim. Biophys Acta 1339 (1997) 39-52. The numbering system used inthis reference forms the basis of the numbering system used in thepresent application.

With respect to the possible change of the amino acid residuecorresponding to residue 214 in Photinus pyralis luciferase, the polaramino acid threonine is suitably replaced with a non polar amino acidsuch as alanine, glycine, valine, lecine, isoleucine, proline,phenylalanine, methionine, tryptophan or cysteine. A particularlypreferred substitution for the threonine residue corresponding toresidue 214 in Photinus pyralis is alanine. A more preferredsubstitution is cysteine. However, different polar residues such asasparagine at this position may also enhance the thermostability of thecorresponding enzyme having threonine at this position.

Other amino acids which appear at this position in wild-type luciferaseenzymes include glycine (Luciola mingrelica, Hotaria paroula),asparagine (Pyrophorus plagiophthalamus, GR, YC, YE and OR, Luciolacruciata, Luciola lateralis, Lampyris noctiluca, Pyrocelia nayakoPhotinus pennsylanvanica LY, J29) and serine (position 211 inPhrixothrix luciferase). These may advantageously be substituted withnon-polar or different non-polar side chains such as alanine andcysteine.

As regards the possible change of the amino acid residue correspondingto residue 232 in Photinus pyralis luciferase, the nonpolar amino acidisoleucine is suitably replaced with a different non polar amino acidsuch as alanine, glycine, valine, leucine, proline, phenylalanine,methionine, tryptophan or cysteine. Other amino acids appearing at thisposition in wild type sequences include serine and asparagine (as wellas valine or alanine at corresponding position 229 in Phritothix greenand red respectively). Suitably, these polar residues are substituted bynon-polar residues such as those outlined above. A particularlypreferred substitution for the residue corresponding to residue 232 inPhotinus pyralis luciferase and to residue 234 of Luciola mingrelica,Luciola cruciata or Luciola lateralis luciferase is alanine, where thisrepresents a change of amino acid over the wild-type sequence. Changesof the amino acid residue corresponding to residue 295 in Photinuspyralis luciferase and to residue 297 of Luciola mingrelica, Luciolacruciata or Luciola lateralis luciferase, may also affect thethermostability of the protein. (This corresponds to position 292 inPhrixothix luciferase.) In general, the amino acid at this position is anon-polar amino acid phenylalanine or leucine. These are suitablychanged for different non-polar amino acids. For example, in Photinuspyralis, the non-polar amino acid phenylalanine is suitably replacedwith a different non polar amino acid, such as alanine, leucine,glycine, valine, isoleucine, proline, methionine, tryptophan orcysteine. A particularly preferred substitution for the phenylalanineresidue corresponding to residue 214 in Photinus pyralis luciferase isleucine.

Mutation at the amino acid residue corresponding to amino acid 14 of thePhotinus pyralis luciferase or to amino acid 16 in Luciola luciferase,(13 in Phrixothrix luciferase) is also possible. This amino acid residue(which is usually phenylalanine, but may also be leucine, serine,arginine or in some instances tyrosine) is suitably changed to adifferent amino acid, in particular to a different nonpolar amino acidsuch as alanine, valine, leucine, isoleucine, proline, methionine ortryptophan, preferably alanine.

Mutation at the amino acid residue corresponding to amino acid 35 of thePhotinus pyralis luciferase or to amino acid residue 37 in Luciolamingrelica luciferase (corresponding to amino acid 38 in other Luciolaspp. And in Phrixothrix) may also be effective. This amino acid variesamongst wild type enzymes, which may include leucine (Photinus pyralis)but also lysine, histidine, glycine, alanine, glutamine and asparticacid at this position. Suitably the amino residue at this position issubstituted with a non-polar amino acid residue or a different non-polaramino acid such as

such as alanine, valine, phenylalanine, isoleucine, proline, methionineor tryptophan. A preferred amino acid at this position is alanine, wherethis is different to the wild-type enzyme.

Mutations at the amino acid corresponding to position 14 of the Photinuspyralis sequence and/or mutation at the amino acid residue correspondingto amino acid 35 of the Photinus pyralis luciferase are preferably notthe only mutation in the enzyme.

They are suitably accompanied by others of the mutations defined above,in particular those at positions corresponding to positions 214, 395 or232 of Photinus pyralis luciferase.

Changes of the amino acid residue corresponding to residue 105 inPhotinus pyralis luciferase and to residue 106 of Luciola mingrelica,Luciola cruciata or Luciola lateralis luciferase, (102 in Phrixothrix)may also affect the thermostability of the protein. In general, theamino acid at this position is a non-polar amino acid alanine orglycine, or serine in Phrixothrix. These are suitably changed fordifferent non-polar amino acids. For example, in Photinus pyralis, thenon-polar amino acid alanine is suitably replaced with a different nonpolar amino acid, such as phenylalanine, leucine, glycine, valine,isoleucine, proline, methionine or tryptophan. A particularly preferredsubstitution for the alanine residue corresponding to residue 105 inPhotinus pyralis luciferase is valine.

Changes of the amino acid residue corresponding to residue 234 inPhotinus pyralis luciferase and to residue 236 of Luciola mingrelica,Luciola cruciata or Luciola lateralis luciferase (231 in Phrixothrix),may also affect the thermostability of the protein. In general, theamino acid at this position is aspartic acid or glycine and in somecases, glutamine or threonine. These are suitably changed for non-polaror different non-polar amino acids as appropriate. For example, inPhotinus pyralis, the amino acid residue is aspartic acid is suitablyreplaced with a non polar amino acid, such as alanine, leucine, glycine,valine, isoleucine, proline, methionine or tryptophan. A particularlypreferred substitution for the phenylalanine residue corresponding toresidue 234 in Photinus pyralis luciferase is glycine. Where a non-polaramino acid residue such as glycine is present at this position (forexample in Luciola luciferase), this may be substituted with a differentnon-polar amino acid.

Changes of the amino acid residue corresponding to residue 420 inPhotinus pyralis luciferase and to residue 422 of Luciola mingrelica,Luciola cruciata or Luciola lateralis luciferase (417 in Phrixothrixgreen and 418 in Phrixothrix red), may also affect the thermostabilityof the protein. In general, the amino acid at this position is anuncharged polar amino acid serine or threonine or glycine. These aresuitably changed for different uncharged polar amino acids. For example,in Photinus pyralis, the serine may be replaced with asparagine,glutamine, threonine or tyrosine, and in particular threonine.

Changes of the amino acid residue corresponding to residue 310 inPhotinus pyralis luciferase and to residue 312 of Luciola mingrelica,Luciola cruciata or Luciola lateralis luciferase, may also affect thethermostability of the protein. The amino acid residue at this positionvaries amongst the known luciferase proteins, being histidine inPhotinus pyralis, Pyrocelia nayalco, Lampyris noctiluca and some formsof Photinus pennsylanvanica luciferase, threonine in Luciola mingrelica,Hotaria paroula and Phrixothix (where it is amino acid 307) luciferase,valine in Luciola cruciata and Luciola lateralis, and asparagine in somePyrophorus plagiophthalamus luciferase. Thus, in general, the amino acidat this position is hydrophilic amino acid which may be changed for adifferent amino acid residue which increases thermostability of theenzyme. A particularly preferred substitution for the histidine residuecorresponding to residue 310 in Photinus pyralis luciferase is arginine.

Other mutations may also be present in the enzyme. For example, in apreferred embodiment, the protein also has the amino acid at positioncorresponding to amino acid 354 of the Photinus pyralis luciferase (356in Luciola luciferase and 351 in Phrixothrix) changed from glutamate, inparticular to an amino acid other than glycine, proline or asparticacid. Suitably, the amino acid at this position is tryptophan, valine,leucine, isoleucine are asparagine, but most preferably is lysine orarginine. This mutation is described in WO 95/25798.

In an alternative preferred embodiment, the protein also has the aminoacid at the position corresponding to amino acid 217 in Luciolaluciferase (215 in Photinus pyralis) changed to a hydrophobic amino acidin particular to isoleucine, leucine or valine as described inEP-A-052448.

The proteins may contain further mutations in the sequence provided theluciferase activity of the protein is not unduly compromised. Themutations suitably enhance the properties of the enzyme or better suitit for the intended purpose in some way. This may mean that they resultin enhanced thermostability and/or colour shift properties, and/or theK_(m) for ATP of the enzymes. Examples of mutations which give rise tocolour shifts are described in WO95/18853. Mutations which affect K_(m)values are described for example in WO 96/22376 and International PatentApplication No. PCT/GB98/01026 which are incorporated herein byreference.

Proteins of the invention suitably have more than one such mutation, andpreferably all three of the mutations described above.

Proteins of the invention include both wild-type and recombinantluciferase enzymes. They have at least 60% similarity to the sequencesof Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciolalateralis or other luciferase enzymes as discussed above in the sensethat at least 60% of the amino acids present in the wild-type enzymesare present in the proteins of the invention. Such proteins can have agreater degree of similarity, in particular at least 70%, morepreferably at least 80% and most preferably at least 90% to thewild-type enzymes listed above. Similar proteins of this type includeallelic variants, proteins from other insect species as well asrecombinantly produced enzymes.

They may be identified for example, in that they are encoded by nucleicacids which hybridise with sequences which encode wild-type enzymesunder stringent hybridisation conditions, preferably high stringencyconditions. Such conditions would be well understood by the personskilled in the art, and are exemplified for example in Sambrook et al.(1989) Molecular Cloning, Cold Spring Harbor Laboratory Press). Ingeneral terms, low stringency conditions can be defined as 3×SCC atabout ambient temperature to about 65° C., and high stringencyconditions as 0.1×SSC at about 65° C. SSC is the name of a buffer of0.15M NaCl, 0.015M trisodium citrate. 3×SSC is three times as strong asSSC and so on.

In particular, the similarity of a particular sequence to the sequencesof the invention may be assessed using the multiple alignment methoddescribed by Lipman and Pearson, (Lipman, D. J. & Pearson, W. R. (1985)Rapid and Sensitive Protein Similarity Searches, Science, vol 227, pp1435-1441). The “optimised” percentage score should be calculated withthe following parameters for the Lipman-Pearson algorithm:ktup=1, gappenalty=4 and gap penalty length=12. The sequence for which similarityis to be assessed should be used as the “test sequence” which means thatthe base sequence for the comparison, such as the sequence of Photinuspyralis or any of the other sequences listed above, as recorded in Ye etal., supra., or in the case of Phrixotrix, as described in Biochemistry,1999, 38, 8271-8279, should be entered first into the algorithm.Generally, Photinus pyralis will be used as the reference sequence.

Particular examples of proteins of the invention are wild-typeluciferase sequence with the mutations as outlined above. The proteinshave at least one and preferably more than one such mutation.

The invention further provides nucleic acids which encode theluciferases as described above. Suitably, the nucleic acids are basedupon wild-type sequences which are well known in the art. Suitablemutation to effect the desired mutation in the amino acid sequence wouldbe readily apparent, based upon a knowledge of the genetic code.

The nucleic acids of the invention are suitably incorporated into anexpression vector such as a plasmid under the control of controlelements such as promoters, enhancers, terminators etc. These vectorscan then be used to transform a host cell, for example a prokaryotic oreukaryotic cell such as a plant or animal cell, but in particular aprokaryotic cell such as E. coli so that the cell expresses the desiredluciferase enzyme. Culture of the thus transformed cells usingconditions which are well known in the art will result in the productionof the luciferase enzyme which can then be separated from the culturemedium. Where the cells are plant or animal cells, plants or animals maybe propagated from said cells. The protein may then be extracted fromthe plants, or in the case of transgenic animals, the proteins may berecovered from milk. Vectors, transformed cells, transgenic plants andanimals and methods of producing enzyme by culturing these cells allform further aspects of the invention.

The Photinus pyralis T214A mutant luciferase was created by randommutagenesis as described hereinafter. It was found that the T214A singlepoint mutation has greater thermostability than wild type luciferase.

Two new triple mutant luciferases: E354K/T214A/A215L andE354K/T214A/I232A were also prepared and these also have exhibitedgreater thermostability.

Particular examples of mutant enzymes of Photinus pyralis which fallwithin the scope of the invention include the following:

I232A/E354K T214A/I232A/E354K A215L/I232A/E354K T214A/I232A/E354K/A215LI232A/E354K/T214A/F295L I232A/E354K/T214A F295L/F14A/L35AI232A/E354K/T214A/F295L/F14A/L35A/A215L A105V T214A T214C T214N T295LI232A F14A L35A D234G S420T H310R

or equivalents of any of these when derived from the luciferases ofother species.

The mutations for the creation of the triple mutant were introduced tothe luciferase gene on plasmid pET23 by site-directed mutagenesis,(PCR). The oligonucleotides added to the PCR reaction in order to effectthe relevant mutations are given in the Examples below.

It has been reported previously that the effect of point mutations atthe 354 and 215 positions are additive. This invention provides thepossibility of combining three or more such mutations to provide stillgreater thermostability.

Thermostable luciferase of the invention will advantageously be employedin any bioluminescent assay which utilises the luciferase/luciferinreaction as a signalling means. There are many such assays known in theliterature. The proteins may therefore be included in kits prepared witha view to performing such assays, optionally with luciferin and anyother reagents required to perform the particular assay.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be particularly described by way of example withreference to the accompanying diagrammatic drawings in which:

FIGS. 1A and 1B illustrate the plasmids used in the production ofmutants in accordance with the invention;

FIG. 2 shows the results of heat inactivation studies on luciferasesincluding luciferases of the invention;

FIGS. 3A-3H show the results of thermostability experiments on variousluciferase mutants;

FIG. 4 shows the results of thermostability experiments on otherluciferase mutants; and

FIG. 5 shows ologonucleotides (SEQ ID NOs: 1-10, 11/36 and 12-33) usedin the preparation of mutant enzymes of the invention.

EXAMPLE 1 Identification of Thermostable Mutant Luciferase

The error-prone PCR was based on the protocol devised by Fromant et al.,Analytical Biochemistry, 224, 347-353 (1995).

The dNTP mix in this reaction was:

35 mM dTTP12.5 mM dGTP22.5 mM dCTP14 mM dATP

The PCR conditions were:

0.5 μl (50 ng) plasmid pPW601a J54*5.0 μl 10×KC1 reaction buffer1 μl each of W56 and W57⁺ (60 pmoles of each primer)1 μl BIOTAQ (thermostable) DNA polymerase (5U)2 μl dNTPs (see above)1.76 μl MgCl₂ (50 mM stock)1 μl mNCl₂ (25 mM stock) [final concentration in reaction=3.26 mM]36.7 μl dH₂O*Plasmid pPW601aJ54 is a mutated version of pPW601a (WO 95/25798) wherean NdeI site has been created within the 3 bases prior to the ATG startcodon. This allows for easy cloning from pPW601a into the pET23 vector.

+Primer Sequences:

W56: (SEQ ID NO: 34) 5′ - AAACAGGGACCCATATGGAAGACGC - 3′ W57:(SEQ ID NO: 35) 5′ - AATTAACTCGAGGAATTTCGTCATCGCTGAATACAG - 3′)Cycling parameters were:

94° C.-5 min

Then 12× cycles of: 94° C.-30s

-   -   55° C.-30s    -   72° C.-5 min    -   72° C.-10 min

The PCR products were purified from the reaction mix using a ClontechADVANTAGE PCR-pure kit. An aliquot of the purified products was thendigested with the restriction enzymes NdeI and XhoI. The digested PCRproducts were then “cleaned up” with the ADVANTAGE kit and ligated intothe vector pET23a which had been digested with the same enzymes.

Ligation Conditions:

4 μl pET23a (56 ng)5 μl PCR products (200 ng)3 μl 5× Gibco BRL ligase reaction buffer1 μl Gibco BRL ligase (10U)2 μl dH₂O

The ligation was carried out overnight at 16° C.

The ligated DNAs were then purified using the ADVANTAGE kit and thenelectroporated into electrocompetent E. coli HB101 cells (1 mm cuvettes,1.8 Kv).

Eleven electroporations were performed and the cells were then added to40 ml of TY broth containing 50 μg/ml ampicillin. The cells were thengrown overnight at 37° C. The entire 50 ml of culture grown overnightwas used to purify plasmid DNA. This is the library.

Screening the Library

An aliquot of the plasmid library was used to electroporate E. coli BL21DE3 cells. These cells were then plated onto LB agar containing 50 μg/mlampicillin and grown overnight at 37° C.

The next day, colonies were picked and patched onto nylon filters on LBagar+amp plates and growth continued overnight at 37° C. The next day,filters were overlaid with a solution of luciferin—500 μM in 100 mMsodium citrate pH5.0. The patches were then viewed in a darkroom. Onecolony/patch was picked from 200 for further analysis.

Characterisation of the Thermostable Mutant

The E. coli clone harbouring the mutant plasmid was isolated. PlasmidDNA was prepared for ABI sequencing. The entire open reading frameencoding luciferase was sequenced using 4 different oligonucleotideprimers. Sequencing revealed a single point mutation at nt 640 (A→G).Giving a codon change of ACT (T) to GCT (A) at amino acid position 214.

EXAMPLE 2 Preparation of Triple Mutant Enzyme

A mutagenic oligonucleotide was then used to create this same mutationin pMOD1 (A215L/E354K) to create a triple mutant pMOD2(A215L/E354K/T214A). This mutation also creates a unique SacI/SstI sitein pMOD1.

EXAMPLE 3 Preparation of Further Triple Mutant Enzyme

The following primers were used to create the triple mutantT214A/I232A/E354K using a standard PCR reaction and with the pET23plasmid with the T214A mutation as template:

CTGATTACACCCAAGGGGGATG (SEQ ID NO: 26) E354K-senseCATCCCCCTTGGGTGTAATCAG (SEQ ID NO: 27) E354K-antisenseGCAATCAAATCGCTCCGGATACTGC (SEQ ID NO: 30) I232A-senseGCAGTATCCGGAGCGATTTGATTGC (SEQ ID NO: 31) I232A-antisense

EXAMPLE 4 Identification of Thermostable 295 Mutant

The F295 mutant was created using the error-prone PCR method describedby Fromant et al., Analytical Biochemistry, vol 224, 347-353 (1995). ThePCR conditions used were as follows:

0.5 μl (50 ng) plasmid pET235.0 μl 10×KCI reaction buffer1 μl primer 1-60 pmoles of each primer1 μl primer 21 μl BIOTAQ (thermostable)DNA polymerase (5U)2 μl dNTPs, in mixture 35 mM dTTP, 12.5 mM dGTP, 22.5 mM dCTP,14 mM dATP1.76 μl MgCl₂ (50 mM stock)1 μl MnCl₂ (25 mM stock) [final concentration in reaction=3.26 mM]36.7 μl dH₂O

Primer 1 = (SEQ ID NO: 34) 5′ - AAACAGGGACCCATATGGAAGACGC - 3′Primer 2 = (SEQ ID NO: 35) 5′- AATTAACTCGAGGAATTTCGTCATCGCTGAATACAG - 3′

The cycling parameters were:

94° C. for 5 min

15 cycles of: 30 s@ 94° C.

-   -   30 s@ 55° C.    -   5 Min@ 72° C.        then 10 min at 72° C.

The PCR products were purified from the reaction mix using a ClontechADVANTAGE PCR-Pure kit. An aliquot of the purified products was thendigested with the restriction enzymes Ndel and Xhol. The digested PCRproducts were then “cleaned up” with the ADVANTAGE kit and ligated intothe vector pET23a, which had been digested with the same enzymes.

The ligation conditions were as follows:

56 ng pET23a200 ng PCR products3 μl 5× Gibco BRL ligase reaction buffer1 μl Gibco BRL ligase (10U)volume made up to 10 μl with dH₂O

The ligation was carried out overnight at 16° C.

The ligated DNAs were then purified using the Advantage™ kit and thenelectroporated into electrocompetent Escherichia coli DH5α cells (1 mmcuvettes, 1.8 kV). 1 ml of SOC broth was added to each electroporationand the cells allowed to recover and express antibiotic resistance genesencoded by the plasmid. Aliquots of the library were inoculated onto LBagar containing 50 μg/ml ampicillin and the bacteria were grownovernight at 37° C. Nylon filter discs were then overlaid onto the agarplates and the colonies transferred to fresh plates. The original plateswere left at room temperature for the colonies to re-grow. The plateswith the nylon filters were incubated at 42° C. for 2 h before plateswere sprayed with 500 μM luciferin in 100 mM citrate buffer pH5.0 andviewed in a darkroom.

Three thermostable colonies were selected on the basis that they stillglowed after 2 h at 42° C. Plasmid DNA was isolated from these clonesand sequenced, and this revealed the F295L mutation in each case.

EXAMPLE 5

Other mutants of the invention were produced by PCR using appropriatecombinations of the oligonucleotides listed above as well as thefollowing:

GAAAGGCCCGGCACCAGCCTATCCTCTAGAGG (SEQ ID NO: 5) F14A-senseCCTCTAGCGGATAGGCTGGTGCCGGGCCTTTC (SEQ ID NO: 6) F14A-antisenseGAGATACGCCGCGGTTCCTGG (SEQ ID NO: 9) L35A-sense CCAGGAACCGCGGCGTATCTC(SEQ ID NO: 10) L35A-antisense

EXAMPLE 6 Purification of Luciferase and Heat Inactivation Studies

Cells expressing the recombinant mutant luciferases were cultured,disrupted and extracted as described in WO 95/25798 to yield cell freeextracts of luciferase.

Eppendorf tubes containing the cell free extracts were incubatedgenerally at 40° C. unless otherwise stated. Purified preparations ofwild type luciferases (for comparative purposes were incubated inthermostability buffer comprising 50 mM potassium phosphate buffer pH7.8containing 10% saturated ammonium sulphate, 1 mM dithiothreitol and 0.2%bovine serum albumin (BSA). At set times a tube was removed and cooledin an ice/water bath prior to assay with remaining assayed activitybeing calculated as a percentage of the initial activity or relativebioluminesce.

The results are illustrated in FIGS. 2 and 3 hereinafter. It can be seenfrom FIG. 2 that luciferase mutants of the invention have improvedthermostability compared with the previously known mutants.

The dramatic increase in stability over wild-type luciferase (RWT) isclear from FIG. 3.

EXAMPLE 7 Investigations into the Activity of 214 Mutants

A library of 214 mutants was prepared using site-directed mutagenesisusing cassette oligos (FIG. 5) and thermostable mutants selected andtested as described in Example 1. Three particularly thermostablemutants were characterised by sequencing as described in Example 1 asT214A, T214C and T214N.

O/N cultures of E. coli XL1-Blue harbouring plasmids encoding T214,T214A, T214C and T214N were lysed using the Promega lysis buffer. 50 μlof liquid extracts were then heat inactivated at 37° C. and 40° C. overvarious time points. Aliquots 10 μl of heated extract were then testedin the Promega live assay buffer (100 μl).

The results are shown in the following Tables

0 4 min 8 min 22 min (37° C.) rwt T214 11074 5561 2555 343 RLU T214C106449 92471 90515 78816 RLU T214A 63829 52017 45864 35889 RLU T214N60679 49144 41736 29488 RLU % remaining activity 37° C. rwt T214 10050.2 23.1 3.1 T214C 100 86.9 85.0 74.0 T214A 100 81.5 71.8 56.2 T214N100 81.0 68.8 48.6

The experiment was repeated at 40° C. with the 3 mutants

0 4 min 8 min 16 min T214C 104830 79365 72088 56863 RLU T214A 6418743521 28691 14547 RLU T214N 60938 38359 25100 12835 RLU % remainingactivity 40° C. 0 4 min 8 min 16 min T214C 100 73.7 68.8 54.2 T214A 10067.8 44.7 22.7 T214N 100 63.0 41.2 21.1

These results indicate that T214C is significantly more thermostablethan either r-wt or T214A or N. This change in properties is unexpectedas it is usually expected that the more cysteine residues that arepresent, the worse the thermostability.

EXAMPLE 8 Investigation of Other Point Mutations

A series of other Photinus pyralis mutants with single point mutationswere prepared using random error-prone PCR (FIG. 5). Following,screening and sequencing of the mutants generated, the sequencing waschecked using site-directed mutagenesis followed by further sequencing.These were D234G, A105V and F295L. The thermostability of these mutantsas well as recombinant wild-type Photinus pyralis luciferase was tested.Protein samples in Promega lysis buffer were incubated at 37° C. for 10minutes and their activity assayed after 2, 5 and 10 minutes. Theresults, showing that each mutation produced enhanced thermostabilityover wild type, is shown in FIG. 4.

1. An isolated recombinant luciferase having luciferase activity and anamino acid sequence which differs from wild-type luciferase fromPhotinus pyralis, Luciola mingrelica, Luciola cruciata, Luciolalateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyrisnoctiluca, Pyrocoelia miyako or Photinus pennsylvanica, in that in thesequence of the recombinant luciferase, the amino acid residuecorresponding to phenylalanine 295 in Photinus pyralis wild-typeluciferase or to leucine 297 in Luciola mingrelica, Luciola cruciata orLuciola lateralis wild-type luciferases, is mutated as compared to thecorresponding amino acid which appears in the corresponding wild-typeluciferase sequence, such that the recombinant luciferase has increasedthermostability as compared to the corresponding wild-type luciferase.2. The recombinant luciferase of claim 1, wherein the recombinantluciferase is a mutated form of Photinus pyralis, Luciola mingrelica,Luciola cruciata, or Luciola lateralis wild-type luciferase.
 3. Therecombinant luciferase of claim 2, wherein the recombinant luciferase isa mutated form of Photinus pyralis wild-type luciferase and wherein theamino acid residue corresponding to position 295 of Photinus pyraliswild-type luciferase is leucine.
 4. The recombinant luciferase of claim2, further comprising an amino acid other than phenylalanine at theresidue corresponding to position 14 of Photinus pyralis wild-typeluciferase, at the residue corresponding to position 16 of Luciolamingrelica wild-type luciferase, or at the residue corresponding toposition 17 of Luciola cruciata or Luciola lateralis wild-typeluciferase.
 5. The recombinant luciferase of claim 4, wherein the aminoacid at the residue corresponding to position 14 of Photinus pyraliswild-type luciferase, at the residue corresponding to position 16 ofLuciola mingrelica wild-type luciferase, or at the residue correspondingto position 17 of Luciola cruciata or Luciola lateralis wild-typeluciferase is alanine.
 6. The recombinant luciferase of claim 2, furthercomprising an amino acid other than leucine at the residue correspondingto position 35 of Photinus pyralis wild-type luciferase, or lysine atthe residue corresponding to position 37 of Luciola mingrelica wild-typeluciferase or at the residue corresponding to position 38 of Luciolacruciata or Luciola lateralis wild-type luciferase.
 7. The recombinantluciferase of claim 6, wherein the amino acid at the residuecorresponding to position 35 of Photinus pyralis wild-type luciferase,at the residue corresponding to position 37 of Luciola mingrelicawild-type luciferase, or at the residue corresponding to position 38 ofLuciola cruciata or Luciola lateralis wild-type luciferase is alanine.8. The recombinant luciferase of claim 2, further comprising an aminoacid residue other than glutamic acid at the amino acid residuecorresponding to position 354 of Photinus pyralis wild-type luciferaseor at the amino acid residue corresponding to position 356 of Luciolamingrelica, Luciola cruciata or Luciola lateralis wild-type luciferase.9. The recombinant luciferase of claim 8, wherein the amino acid at theresidue corresponding to position 354 of Photinus pyralis wild-typeluciferase or at the amino acid residue corresponding to position 356 ofLuciola mingrelica, Luciola cruciata or Luciola lateralis wild-typeluciferase is lysine.
 10. The recombinant luciferase of claim 2, furthercomprising an amino acid residue other than alanine at the amino acidresidue corresponding to position 215 of Photinus pyralis wild-typeluciferase or at the amino acid residue corresponding to position 217 ofLuciola mingrelica or Luciola lateralis wild-type luciferase, orthreonine at the amino acid residue corresponding to position 217 ofLuciola cruciata wild-type luciferase.
 11. The recombinant luciferase ofclaim 10, wherein the amino acid at the residue corresponding toposition 215 of Photinus pyralis wild-type luciferase or at the aminoacid residue corresponding to position 217 of Luciola mingrelica,Luciola lateralis or Luciola cruciata wild-type luciferase is lysine.12. The recombinant luciferase of claim 3, wherein the amino acidresidue corresponding to position 214 is alanine, position 232 isalanine and position 354 is lysine.
 13. The recombinant luciferase ofclaim 12, wherein the amino acid residue corresponding to position 14 isalanine and position 35 is alanine.
 14. The recombinant luciferase ofclaim 13, wherein the amino acid residue corresponding to position 215is leucine.
 15. An isolated recombinant luciferase generated bysubstituting an amino acid residue corresponding to phenylalanine 295 inPhotinus pyralis wild-type luciferase, or to leucine 297 of Luciolamingrelica, Luciola cruciata or Luciola lateralis wild-type luciferaseswith a different amino acid, wherein the wild-type luciferase is fromPhotinus pyralis, Luciola mingrelica, Luciola cruciata, Luciolalateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyrisnoctiluca, Pyrocoelia miyako or Photinus pennsylvanica, and wherein therecombinant luciferase has increased thermostability as compared to thecorresponding wild-type luciferase.
 16. The recombinant luciferase ofclaim 15, wherein the recombinant luciferase is a mutated form ofPhotinus pyralis wild-type luciferase and wherein the amino acid residuecorresponding to position 295 of Photinus pyralis wild-type luciferaseis leucine.
 17. An isolated recombinant luciferase having luciferaseactivity and an amino acid sequence which differs from wild-typeluciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata,Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus,Lampyris noctiluca, Pyrocoelia miyako or Photinus pennsylvanica,generated by substituting an amino acid residue corresponding tophenylalanine 295 in Photinus pyralis wild-type luciferase, or toleucine 297 of Luciola mingrelica, Luciola cruciata or Luciola lateraliswild-type luciferases, with a different amino acid, wherein thewild-type luciferase is from Photinus pyralis, Luciola mingrelica,Luciola cruciata, Luciola lateralis, Hotaria parvula, Pyrophorusplagiophthalamus, Lampyris noctiluca, Pyrocoelia miyako or Photinuspennsylvanica, and wherein the recombinant luciferase has increasedthermostability as compared to the corresponding wild-type luciferase.18. The recombinant luciferase of claim 17, wherein the recombinantluciferase is a mutated form of Photinus pyralis wild-type luciferaseand wherein the amino acid residue corresponding to position 295 ofPhotinus pyralis wild-type luciferase is leucine.
 19. An isolatednucleic acid which encodes the recombinant luciferase of claim
 1. 20. Avector comprising the nucleic acid of claim
 19. 21. An isolated celltransformed with the vector of claim
 20. 22. The cell of claim 21 whichis a prokaryotic cell.
 23. A bioluminescent assay comprising the stepsof contacting the recombinant luciferase of claim 1 with luciferin anddetecting bioluminescence.
 24. A kit comprising the recombinantluciferase of claim 1.